RESUMEN
The genomes of myxobacteria harbor a variety of biosynthetic gene clusters encoding numerous secondary metabolites, including ribosomally synthesized and post-translationally modified peptides (RiPPs) with diverse chemical structures and biological activities. However, the biosynthetic potential of RiPPs from myxobacteria remains barely explored. Herein, we report a novel myxobacteria lanthipeptide myxococin identified from Myxococcus fulvus. Myxococins represent the first example of lanthipeptides, of which the characteristic multiple thioether rings are installed by employing a Class II lanthipeptide synthetase MfuM and a Class I lanthipeptide cyclase MfuC in a cascaded way. Unprecedentedly, we biochemically characterized the first M61 family aminopeptidase MfuP involved in RiPP biosynthesis, demonstrating that MfuP showed the activity of an endopeptidase activity. MfuP is leader-independent but strictly selective for the multibridge structure of myxococin A and responsible for unwrapping two rings via amide bond hydrolysis, yielding myxococin B. Furthermore, the X-ray crystal structure of MfuP and structural analysis, including active-site mutations, are reported. Finally, myxococins are evaluated to exhibit anti-inflammatory activity in lipopolysaccharide-induced macrophages without detectable cytotoxicity.
Asunto(s)
Myxococcales , Péptidos/química , Procesamiento Proteico-PostraduccionalRESUMEN
A characteristic feature of COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the dysregulated immune response with impaired type I and III interferon (IFN) expression and an overwhelming inflammatory cytokine storm. RIG-I-like receptors (RLRs) and cGAS-STING signaling pathways are responsible for sensing viral infection and inducing IFN production to combat invading viruses. Multiple proteins of SARS-CoV-2 have been reported to modulate the RLR signaling pathways to achieve immune evasion. Although SARS-CoV-2 infection also activates the cGAS-STING signaling by stimulating micronuclei formation during the process of syncytia, whether SARS-CoV-2 modulates the cGAS-STING pathway requires further investigation. Here, we screened 29 SARS-CoV-2-encoded viral proteins to explore the viral proteins that affect the cGAS-STING signaling pathway and found that SARS-CoV-2 open reading frame 10 (ORF10) targets STING to antagonize IFN activation. Overexpression of ORF10 inhibits cGAS-STING-induced interferon regulatory factor 3 phosphorylation, translocation, and subsequent IFN induction. Mechanistically, ORF10 interacts with STING, attenuates the STING-TBK1 association, and impairs STING oligomerization and aggregation and STING-mediated autophagy; ORF10 also prevents the endoplasmic reticulum (ER)-to-Golgi trafficking of STING by anchoring STING in the ER. Taken together, these findings suggest that SARS-CoV-2 ORF10 impairs the cGAS-STING signaling by blocking the translocation of STING and the interaction between STING and TBK1 to antagonize innate antiviral immunity.
Asunto(s)
COVID-19 , Interferón Tipo I , Autofagia , Humanos , Inmunidad Innata , Interferón Tipo I/genética , Interferones , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Sistemas de Lectura Abierta , Proteínas Serina-Treonina Quinasas/genética , SARS-CoV-2 , Proteínas Virales/metabolismoRESUMEN
The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.
Asunto(s)
Proteína 58 DEAD Box/inmunología , Evasión Inmune/genética , Interferones/inmunología , Nucleotidiltransferasas/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2/inmunología , Receptor Toll-Like 3/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Animales , Chlorocebus aethiops , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteína 58 DEAD Box/genética , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/inmunología , Interferones/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Nucleotidiltransferasas/genética , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Plásmidos/química , Plásmidos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Receptores Inmunológicos/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 3/genética , Transfección , Células Vero , Replicación Viral/inmunologíaRESUMEN
The ongoing outbreak of a new coronavirus (2019-nCoV, or severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) has caused an epidemic of the acute respiratory syndrome known as coronavirus disease (COVID-19) in humans. SARS-CoV-2 rapidly spread to multiple regions of China and multiple other countries, posing a serious threat to public health. The spike (S) proteins of SARS-CoV-1 and SARS-CoV-2 may use the same host cellular receptor, angiotensin-converting enzyme 2 (ACE2), for entering host cells. The affinity between ACE2 and the SARS-CoV-2 S protein is much higher than that of ACE2 binding to the SARS-CoV S protein, explaining why SARS-CoV-2 seems to be more readily transmitted from human to human. Here, we report that ACE2 can be significantly upregulated after infection of various viruses, including SARS-CoV-1 and SARS-CoV-2, or by the stimulation with inflammatory cytokines such as interferons. We propose that SARS-CoV-2 may positively induce its cellular entry receptor, ACE2, to accelerate its replication and spread; high inflammatory cytokine levels increase ACE2 expression and act as high-risk factors for developing COVID-19, and the infection of other viruses may increase the risk of SARS-CoV-2 infection. Therefore, drugs targeting ACE2 may be developed for the future emerging infectious diseases caused by this cluster of coronaviruses.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , COVID-19/inmunología , Receptores Virales/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Enzima Convertidora de Angiotensina 2/inmunología , COVID-19/virología , Expresión Génica , Células HEK293 , Humanos , Interferones/farmacología , Análisis por Micromatrices , Unión Proteica , Receptores Virales/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Regulación hacia ArribaRESUMEN
Clinical benefit from immunotherapy of B7-H1/PD-1 checkpoint blockade indicates that it is important to understand the regulatory mechanism of B7-H1 expression in cancer cells. As an adaptive response to the endogenous antitumor immunity, B7-H1 expression is up-regulated in HCC cells. B7-H1 expression is induced mainly by IFN-γ released from tumor-infiltrating T cells in HCC. In addition, HCC is a prototype of inflammation-related cancer and TNF-α is a critical component of inflammatory microenvironment of HCC. In the present study, we asked whether TNF-α can promote the expression of B7-H1 induced by IFN-γ in HCC cells. We found that JAK/STAT1/IRF1 was the primary pathway responsible for induction of B7-H1 expression by IFN-γ in human HCC cell lines. TNF-α and IFN-γ synergistically induced the expression of B7-H1 in the HCC cells. Moreover, the mechanism of the synergy was that TNF-α enhanced IFN-γ signaling by upregulating the expression of IFN-γ receptors. Furthermore, B7-H1 expression induced synergistically by TNF-α and IFN-γ in murine HCC cells facilitated tumor growth in vivo. Our findings suggest that TNF-α may enhance the adaptive immune resistance mediated by IFN-γ-induced B7-H1 in HCC cells.
Asunto(s)
Antígeno B7-H1/biosíntesis , Carcinoma Hepatocelular/metabolismo , Interferón gamma/metabolismo , Neoplasias Hepáticas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Femenino , Humanos , Interferón gamma/farmacología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I-MAVS complex to attenuate the RIG-I-mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.
Asunto(s)
Proteasas 3C de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/genética , Proteína 58 DEAD Box/genética , ADN Helicasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas de Unión al ARN/genética , Receptores Inmunológicos/genética , SARS-CoV-2/genética , Gránulos de Estrés/genética , Animales , Chlorocebus aethiops , Proteasas 3C de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteína 58 DEAD Box/inmunología , ADN Helicasas/inmunología , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Evasión Inmune , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Poli I-C/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , Unión Proteica , ARN Helicasas/inmunología , Proteínas con Motivos de Reconocimiento de ARN/inmunología , ARN Bicatenario/genética , ARN Bicatenario/inmunología , Proteínas de Unión al ARN/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Virus Sendai/genética , Virus Sendai/inmunología , Transducción de Señal , Gránulos de Estrés/efectos de los fármacos , Gránulos de Estrés/inmunología , Gránulos de Estrés/virología , Células Vero , Vesiculovirus/genética , Vesiculovirus/inmunologíaRESUMEN
The global epidemic caused by the coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in the infection of over 200 million people. To extend the knowledge of interactions between SARS-CoV-2 and humans, we systematically investigate the interactome of 29 viral proteins in human cells by using an antibody-based TurboID assay. In total, 1,388 high-confidence human proximal proteins with biotinylated sites are identified. Notably, we find that SARS-CoV-2 manipulates the antiviral and immune responses. We validate that the membrane protein ITGB1 associates angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 entry. Moreover, we reveal that SARS-CoV-2 proteins inhibit activation of the interferon pathway through the mitochondrial protein mitochondrial antiviral-signaling protein (MAVS) and the methyltransferase SET domain containing 2, histone lysine methyltransferase (SETD2). We propose 111 potential drugs for the clinical treatment of coronavirus disease 2019 (COVID-19) and identify three compounds that significantly inhibit the replication of SARS-CoV-2. The proximity labeling map of SARS-CoV-2 and humans provides a resource for elucidating the mechanisms of viral infection and developing drugs for COVID-19 treatment.
Asunto(s)
Anticuerpos/inmunología , Antivirales/inmunología , SARS-CoV-2/inmunología , Enzima Convertidora de Angiotensina 2/inmunología , Antivirales/farmacología , COVID-19/inmunología , Humanos , Integrina beta1/inmunología , Pruebas de Sensibilidad Microbiana , Tratamiento Farmacológico de COVID-19RESUMEN
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has quickly spread worldwide and has affected more than 10 million individuals. A typical feature of COVID-19 is the suppression of type I and III interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism by which SARS-CoV-2 evades antiviral immunity remains elusive. Here, we reported that the SARS-CoV-2 membrane (M) protein inhibits the production of type I and III IFNs induced by the cytosolic dsRNA-sensing pathway mediated by RIG-I/MDA-5-MAVS signaling. In addition, the SARS-CoV-2 M protein suppresses type I and III IFN induction stimulated by SeV infection or poly (I:C) transfection. Mechanistically, the SARS-CoV-2 M protein interacts with RIG-I, MAVS, and TBK1, thus preventing the formation of the multiprotein complex containing RIG-I, MAVS, TRAF3, and TBK1 and subsequently impeding the phosphorylation, nuclear translocation, and activation of IRF3. Consequently, ectopic expression of the SARS-CoV-2 M protein facilitates the replication of vesicular stomatitis virus. Taken together, these results indicate that the SARS-CoV-2 M protein antagonizes type I and III IFN production by targeting RIG-I/MDA-5 signaling, which subsequently attenuates antiviral immunity and enhances viral replication. This study provides insight into the interpretation of SARS-CoV-2-induced antiviral immune suppression and illuminates the pathogenic mechanism of COVID-19.
Asunto(s)
COVID-19/metabolismo , Proteína 58 DEAD Box/metabolismo , Interferón Tipo I/biosíntesis , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferones/biosíntesis , SARS-CoV-2/metabolismo , Transducción de Señal , Proteínas de la Matriz Viral/metabolismo , Animales , COVID-19/genética , Chlorocebus aethiops , Proteína 58 DEAD Box/genética , Células HEK293 , Células HeLa , Humanos , Interferón Tipo I/genética , Helicasa Inducida por Interferón IFIH1/genética , Interferones/genética , Receptores Inmunológicos , SARS-CoV-2/genética , Células Vero , Proteínas de la Matriz Viral/genética , Interferón lambdaRESUMEN
The effectiveness of immunotherapy targeting the immune checkpoint PD-L1/PD-1 pathway highlights importance of elucidating the regulatory mechanisms of PD-L1 expression in cancer cells. Previous studies demonstrate that oncogene MYC up-regulates PD-L1 expression in lymphomas. In the present study, we investigated the regulatory role of MYC in the PD-L1 expression induced by IFN-γ in HCC cells. Unexpectedly, knockdown of MYC expression using siRNA assay increased the inducible expression of PD-L1 both at mRNA and protein levels. Mechanistically, the inhibition of MYC elevated expression of STAT1, a critical component of IFN-γ signaling pathway, leading to the elevation of PD-L1 expression in HCC cells exposed to IFN-γ. These results suggest that MYC may down-regulate PD-L1 expression in the context of HCC. This study implicates that a combination therapy targeting MYC function and PD-L1/PD-1 pathway might be effective for treatment of HCC.